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Jun 2, 2014 - Le Poul E, Brezillon S, Tyldesley R, Suarez-Huerta N, Vandeput F, Blanpain C,. Schiffmann SN, Vassart G, Parmentier M: The metastasis ...
Ji et al. BMC Cancer 2014, 14:723 http://www.biomedcentral.com/1471-2407/14/723

RESEARCH ARTICLE

Open Access

Implication of metastasis suppressor gene, Kiss-1 and its receptor Kiss-1R in colorectal cancer Ke Ji1, Lin Ye1, Fiona Ruge1, Rachel Hargest1, Malcolm D Mason2 and Wen G Jiang1*

Abstract Background: Kiss-1 and Kiss-1R have been suggested as a novel pair of metastasis suppressors for several human solid tumours, however, their role in colorectal cancer remains largely unknown. Therefore, the aim of this study was to investigate the role and signal transduction of Kiss-1 and Kiss-1R in colorectal cancer. Methods: Ribozyme transgenes were used to knockdown high expression of Kiss-1 and Kiss-1R in HT115 and HRT18 cells. The stabilized transfected cells were then used to deduce the influence of Kiss-1 and Kiss-1R on the function of colorectal cancer cells by in vitro assays and ECIS assay. The effect of Kiss-1 on MMPs related to tumour metastasis was also deleted by zymography. The mRNA and protein expression of Kiss-1 and Kiss-1R, and their correlation to the clinical outcome in human colorectal cancer were investigated using real-time PCR and IHC respectively. Results: Knocking down Kiss-1 resulted in increased invasion and migration of colorectal cancer cells. Kisspeptin-10 decreased cellular migration of colorectal cancer cells and required ERK signaling as shown during the ECIS based analyses. Reduction of MMP-9 was caused by Kisspeptin-10 and ERK inhibitor, shown by zymography. In human colorectal cancer tissues, the mRNA expression level of Kiss-1 had a negative correlation with Dukes staging, TNM staging, tumour size and lymph node involvement. Reduction of Kiss-1R was also linked to poor prognosis for the patients. Conclusions: The present study has presented evidence that Kiss-1 may be a putative metastasis suppressor molecule in human colorectal cancer. Keywords: Metastasis suppressor gene, Kiss-1, Kiss-1R, GPR54, Colorectal cancer, Migration, Invasion, MMP-9, ERK signal pathway

Background Colorectal cancer (CRC) is the second most commonly diagnosed cancer and is a major cause for mortality and morbidity globally [1]. The Kiss-1 gene was identified as a human melanoma metastasis suppressor gene through the analysis of subtractive hybridization in highly metastatic cell lines as compared to non-metastatic cell lines [2]. The Kiss-1 gene encodes a protein of 145-amino-acids, which is subsequently cleaved into a family of Kisspeptins, including Kisspeptin-10, Kisspeptin-13, Kisspeptin-14, Kisspeptin-54 respectively [3-5]. Its receptor, Kiss-1R, also known as G-

* Correspondence: [email protected] 1 Cardiff University-Peking University Joint Oncology Institute, Metastasis & Angiogenesis Research Group, Cardiff University School of Medicine, Cardiff CF14 4XN, UK Full list of author information is available at the end of the article

protein coupled receptor 54 (GPR54) was first discovered and cloned from rat brain in 1999 [6]. Kiss-1 and Kiss-1R have been suggested as a novel pair of metastasis suppressors in most cancers [5,7-11]. Correlation between decreased expression of Kiss-1 and poor clinical outcomes has been evident in most malignancies that have been investigated. One possible explanation for the role played by Kiss-1 in cancer biology could be extrapolated from the relationship between Kiss-1 and matrix metalloproteinases (MMPs), whose significance in tumour invasion and metastasis formation is well known [12]. However, few studies have specifically shown a role for Kiss-1 in colorectal cancer as yet. In this study, we examined the expression of Kiss-1 and Kiss-1R in human colorectal cancer and analyzed the potential clinical and prognostic implications. After that, we investigated their effect on the function of

© 2014 Ji et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Ji et al. BMC Cancer 2014, 14:723 http://www.biomedcentral.com/1471-2407/14/723

colorectal cancer cells. On the basis of the data from these experiments, Kiss-1 may play a metastasis suppressor role in human colorectal cancer and be linked to the disease progression of patients, by way of aberrant expression and molecular and cellular mechanism (s) that are yet to be identified.

Methods Patients and tissue specimens

Colorectal cancer tissues (n = 94) and normal background tissues (n = 80) were collected immediately after surgery with approval by the South East Wales Local Research Ethics Committee (ref 05/WSE03/92) and patients’ consent. The tissue samples were stored in a deep freezer (−80°C) until further use. Patients were routinely followed after surgery and the median follow up period was 120 months. Tumour tissues and normal tissues (>10 cm away from the tumor margin) were obtained with confirmation by a pathologist. Cell culture

HT115 and HRT18 cancer cell lines were obtained from the European Collection of Animal Cell Culture (ECACC, Salisbury, England, UK). Peptide receptors and inhibitors, antibodies

ERK inhibitorII (FR180204) (Calbiochem, Germany), Kisspeptin-10 (No.2570) (Tocris Bioscience, Bristol, UK) and Kisspeptin-234 (Tocris Bioscience, Bristol, UK). Kisspeptin-10, a short peptide of 10 amino acids, is proteolytically processed from Kiss-1 [13]. Kisspeptin-234 is a Kisspeptin-10/Kiss-1R antagonist, which belongs to Kisspeptin-10 analog. Antibodies to Kiss-1 (SC-101246) and Kiss-1R (SC-48220) were purchased from Santa Cruz Biotechnologies Inc., (Santa Cruz, CA, USA).

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RNA isolation, cDNA synthesis, RT-PCR, Q-PCR and immunohistochemical staining

RNA extraction kits, reverse transcription kits and RTPCR Mix were purchased from Promega (WI, USA) and Bio-Rad (CA, USA). Conventional PCR primers were designed using Beacon Designer (Palo Alto, CA, USA) and synthesized by Invitrogen (Paisley, Scotland, UK). Following the manufacturer’s protocol, total RNA was isolated using TRI reagent (Sigma). The concentration of RNA was detected by a UV spectrophotometer at 260 and 280 nm. cDNA samples were synthesized and the final reaction volume was 20μl. Primers used for RT-PCR are given in Table 1, and GAPDH was used as the house keeping control. Real-time quantitative PCR, based on the Amplifluor™ technology, was used to quantify the level of mRNA expression of Kiss-1 and Kiss-1R from the cDNA samples of coloretal tissues and cells, mentioned above. All colorectal cDNA samples were synchronously examined for Kiss-1 and Kiss-1R along with a set of internal controls. Q-PCR primers (Table 1) were designed using Beacon Design software (PREMIER Biosoft, Palo Alto, CA). Real-time PCR was carried out using an IcyclerIQ™ (Bio-Rad, Hemel Hempstead, UK) with the following cycling conditions: 94°C for 5 min, 80–90 cycles of: 94°C for 10 sec, 55°C for 35 sec and 72°C for 20 sec. Frozen sections of colorectal tumours and adjacent background tissues were sectioned at a thickness of 6 μm using a cryostat . The samples were mounted onto Super Frost Plus microscope slides (Fisher, UK). The samples were fixed in a mixture of 50% acetone and 50% methanol and then air-dried. After rehydration and blocking with 5% horse serum solution, the sections were probed with the appropriate primary antibody and secondary antibodies. Following the instructions, the

Table 1 Primer sequences used for RT-PCR and Q-PCR in this study Genes Kiss-1

Kiss-1R

GAPDH

Sense (5'-'3)

Antisense (5'-'3)

Conventional PCR

TGAACTCACTGGTTTCTTGG

CGAAGGAGTTCCAGTTGTAG

Quantitative PCR

CATTAGAAAAGGTGGCCTCT

ACTGAACCTGACCGTACAGCCCAGGGATTCTAGCTG

Anti-Kiss1 ribozyme1

CTGCAGCTCTCGGGGGGCG GGGACAGCGAGGTCCCCCC TGATGAGTCCGTGAGGA

ACTAGTGCCAGCTGCTACTGCCAGGCTGAGCCGTTT CGTCCTCACGGACT

Anti-Kiss-1 ribozyme2

CTGCAGCACCGCGCCCTGGGGTG CGGGCTGATGAGTCCGTGAGGA

ACTAGTGTCCGCCCCCCACAGCCGCCAGATTTCGTCC TCACGGACT

Conventional PCR

CTTCATGTGCAAGTTCGTC

CACCAGGAACAGCTGGAT

Quantitative PCR

GCTGGTCATCTACGTCATCT

ACTGAACCTGACCGTACACAGCACAGGAGGAAGGTC

Anti-Kiss-1R ribozyme1

CTGCAGTTCCGCATCGGCTTGTGG CGGCACTGATGAGTCCGTGAGGA

ACTAGTTCGCTGGTCATCTACGTCATTTCGTCCTCACGGACT

Anti-Kiss-1R ribozyme2

CTGCAGAGCCTACCCAGATGCTG AGGCTCTGATGAGTCCGTGAGGA

ACTAGTGCCCCGCCTGGCGCTGGCTGTTTCGTCCTCACGGACT

Conventional PCR

GGCTGCTTTTAACTCTGGTA

GACTGTGGTCATGAGTCCTT

Quantitative PCR

CTGAGTACGTCGTGGAGTC

ACTGAACCTGACCGTACACAGAGATGATGACCCTTTTG

Ji et al. BMC Cancer 2014, 14:723 http://www.biomedcentral.com/1471-2407/14/723

avidin-biotin complex (Vector Laboratories, Burlingame, CA, USA) was applied before staining with diaminobenzidine chromogen. Nuclei were counterstained in Mayer’s haematoxylin. Sections from fresh frozen human placenta were used as positive controls. Generation of Kiss-1 and Kiss-1R ribozyme transgenes and stable transfectants

Hammerhead ribozymes targeting Kiss-1 and Kiss-1R were designed using Zuker’s mRNA Fold programme [14] based on the secondary structure of Kiss-1 and Kiss-1R mRNA (Figure 1A and B, respectively). The ribozymes

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were synthesized using touchdown PCR and cloned into the pEF6/V5-His TOPO TA expression plasmid vector (Invitrogen, Paisley, UK) according to the protocol provided. Ribozyme transgenes and empty plasmids were transfected into the two colorectal cell lines HT115 and HRT18 respectively, utilizing an Easyjet Plus electroporator (EquiBio, Kent, United Kingdom). Following selection using blasticidin, verified transfectants which lost the expression of Kiss-1 and Kiss-1R were used in subsequent experiments. During these experiments, cDNA generated from human placenta was used as a positive control.

Figure 1 Genetic modification of Kiss-1 and Kiss-1R expression in colon cancer cells. A and B: Secondary structures of Kiss-1 (A) and Kiss-1R (B) used to design anti-Kiss1 and Kiss-1 ribozymes C and D: Quantitative real time PCR showed Kiss-1 (C) and Kiss-1R (D) mRNA volume of three repeats which was normalized against corresponding internal control (GAPDH). E and F: Confirmation of Kiss-1 (E) and Kiss-1R (F) knockdown in HT115 cells and HRT18 cells using Western blot.

Ji et al. BMC Cancer 2014, 14:723 http://www.biomedcentral.com/1471-2407/14/723

In vitro cell function assays In vitro cell growth assay

Cells were seeded into 96-well plates at 2,500 cells/well, and cultured using normal media (10% fetal calf serum, 0.1% antibiotics). The cells were cultured in triplicate for 1, 3 and 5 days. After incubation the cells were fixed in 4% formalin and stained with 0.5% crystal violet (w/v). The stained crystal violet was then extracted using 10% (v/v) acetic acid, and the absorbance was determined using a spectrophotometer (Bio-Tek, ELx800) at a wavelength of 540 nm. In vitro cell adhesion assay

A 96-well plate was precoated with 5 μg of Matrigel (CollaborativeResearch Products, Bedford, Massachusetts, USA) and allowed to air dry. Following rehydration using serum free media, 40,000 cells were seeded into each well. After 40 minutes of incubation, non-adherent cells were washed off using BSS. The adherent cells were then fixed with 4% formalin and stained using 0.5% crystal violet. The number of adherent cells were counted under a microscope. In vitro invasion assay

Transwell inserts (with 8 μm pore) were precoated with 50 μg of Matrigel and air dried. Following rehydration, 40,000 cells were seeded into each insert. After incubation for three days, cells which had invaded through the matrix and adhered to the other side of the insert were fixed in 4% formalin, and stained with 0.5% (w/v) crystal violet. The number of invaded cells was then counted under a microscope. In vitro wounding assay for cellular migration

Cells were seeded into a 24-well plate at a density of 200,000 per well and allowed to form a monolayer of cells. The monolayer of cells was then scraped to create a wound. Migration of cells at the wound edges were monitored over a period up to 18 hours. Optimas 6.0 motion analysis (Meyer Instruments, Houston, Texas) was used to track the leading edge of cells to measure the distance of migration. Electric cell-substrate impedance sensing (ECIS)

The ECIS system (Ztheta, Applied Biophysics Inc., USA) was used to quantify cell migration as previously reported by Jiang et al. [15]. HT115 cells were prepared for six repeats per group and seeded at 40,000 cells per well in 200 μl of DMEM medium alone or medium supplemented with 200 nM ERK small inhibitor, 300 nM Kisspeptin-10 and 300 nM Kisspeptin-234. Gelatin zymography assay

Cells were counted and 1×106 cells were seeded to a tissue culture flask. After an overnight incubation and 4 hours

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treatment with appropriate peptide receptor or inhibitor (300 nM Kisspeptin-10, 300 nM Kisspeptin-234 or 200 nM ERK inhibitor), the medium was collected. This method is well established within the laboratory, and has previously been published [16]. Data analysis

The relationship between the expression of Kiss-1 and Kiss-1R and tumour grade, TNM staging and nodal status was respectively analyzed using Mann–Whitney U test (Tables 2 and 3). Quantitative data of IHC, in vitro assays, ECIS assay and zymography assay was analyzed using the Student’s test, Kruskal-Wallis and chi-squared test, where appropriate. Survival analysis and multivariate analysis were carried out using the SPSS20 software package. Differences were considered to be statistically significant at p < 0.05.

Results and discussion Kiss-1 and Kiss-1R expression in colorectal adenocarcinoma tissues and the histopathological/clinical characteristics of the disease

Kiss-1 and Kiss-1R expression was analysed in colorectal cancer tissues and adjacent normal tissues using real time PCR (Tables 2 and 3). Decreased levels of Kiss-1 transcript were seen in Dukes B and C tumours compared with Dukes A carcinomas (p < 0.05). The expression level of Kiss-1 decreased as TNM stage progressed, and statistical analysis revealed significant links between TNM stage I and stage III&IV. A significantly decreased expression of Kiss-1R was observed in tumour tissues compared with normal background tissues. Interestingly, Kiss-1R expression in patients undergoing chemo-radio therapy was considerably higher in comparison to that in patients without therapy. A significant difference was also observed in the expression levels between living patients and those who had died. The average copy numbers of Kiss-1 and Kiss-1R transcripts in Dukes B were then employed as the respective thresholds for the survival analysis. KaplanMeier survival analysis demonstrated that patients with a low expression level of Kiss-1 appeared to have similar overall survival and disease free survival compared to patients with high expression of Kiss-1 (p > 0.05). In contrast to Kiss-1, the expression pattern of Kiss-1R revealed that high levels of Kiss-1R transcript are associated with both a poor overall survival (Figure 2C, p = 0.0011) and poor disease free survival (Figure 2D, p = 0.0033). Multivariate analyses have further demonstrated that Tstage and Kiss-1R are independent prognostic factors (p = 0.025 and p = 0.003, respectively) for colorectal related death. Furthermore, TNM staging and Kiss-1R (p = 0.03 and p = 0.012, respectively) are independent prognostic factors for colorectal cancer related incidence (death, recurrence and metastasis) (Table 4).

Ji et al. BMC Cancer 2014, 14:723 http://www.biomedcentral.com/1471-2407/14/723

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Table 2 The correlation of mRNA expression of Kiss-1 and clinical parameters Category

No.

Median

IQR

Normal

80

97

6-2345

Tumour

94

35

1-2108

Paired normal

68

82

6-2344

Paired tumour

68

8

0-1855

Left colon

22

624

173-1968

Right colon

28

719

44-2000

0.899

Transcolon

2

154

N/A

0.958

Rectum

22

673

175-2522

0.907

A

7

5539

848-24856

B

33

780

104-2272

0.043d

C

32

611

190-1221

0.015d

T1

2

8000

N/A

T2

10

1721

789-10369

1

T3

40

639

71-1332

0.226

T4

18

592

215-1600

0.186

Node 0e

39

872

213-2874

Node 1e

16

715

67-1428

0.326

e

Node 2

15

609

200-643

0.045d

I

9

2081

814-20038

II

30

756

122-2473

0.06

III&IV

32

611

190-1221

0.009d

p

Pa

b

T/N

0.3775

b

Paired T/N

0.1672

Location

c

Dukes classification

Tumour stage

0.0381d

Lymph node involvement

TNM staging

Clinical outcome No invasion

50

835

127-2372

Invasion

26

579

161-948

Disease free

35

759

84-2081

Incidence

23

635

217-2187

No recurrence

58

735

89-2373

Local recurrence

7

643

269-3718

No metastasis

50

735

84-2522

Metastasis

19

634.3

213-1463

Alive

36

661

86-1876

Death

22

572

195-1644

0.1907

0.8115

0.604

0.92

0.923

Tumour (T), Normal (N). bTransverse colon. cp < 0.05. dNode 0 stands for no node involvement; Node 1 stands for 1 to 3 lymph nodes close to the bowel found to contain cancer cells; Node 2 stands for more than 3 lymph nodes found to contain cancer cells and further than 3cm away from the main tumor in the bowel or the presence of cancer cells in lymph nodes connected to the main blood vessels around the bowel. a

Immunochemical staining was carried out on a portion of paired normal and tumour tissues (n = 23 pairs). Intense expression of Kiss-1 and Kiss-1R was primarily observed in

the cytoplasm of epithelial cells in adjacent normal tissues compared with cancer cells (p value < 0.05), as shown in Figure 2A and B.

Ji et al. BMC Cancer 2014, 14:723 http://www.biomedcentral.com/1471-2407/14/723

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Table 3 The correlation of mRNA expression of Kiss-1R and clinical parameters Category

No.

Median

IQR

Normal

80

30

1-2406

Tumour

94